This research focuses on oxidative modification of biopolymers, especially of proteins. The resulting covalent modifications have been implicated in important physiologic and pathologic processes. Determination of the actual roles of oxidative modification in these processes requires the identification of specific proteins which are susceptible to modification and the mapping of the sites of modification in those proteins. During this year, four proteins have been studied in detail towards this goal. The iron regulatory protein 2 was shown to be readily oxidized both in vitro and in vivo, and the oxidatively modified protein was rapidly degraded by the proteasome in iron replete cells. Both oxidation and susceptibility to degradative turnover required the presence of a 73 amino acid loop, and the addition of iron. Thus, oxidative modification of this protein occurs physiologically when cells are iron sufficient. Alpha-1-antitrypsin is the most important antiprotease in pulmonary fluid. Insufficient functional alpha-1-antitrypsin is thought to cause emphysema. Exposure of the antiprotease to oxidizing conditions caused loss of activity, and this was shown to be the consequence of oxidation of either of two methionine residues located at the active site of the protein. Alpha-2-macroglobulin is one of the most important circulating antiproteases. It can be inactivated by exposure to neutrophils or simply to chloramines which are produced by activated neutrophils. We had previously found that inactivation is directly proportional to the fraction of a single tryptophan which was oxidized. That residue has now been identified, and site specific mutants are under construction. Glutamine synthetase has been the subject of many studies of oxidative modification of proteins. Exposure to hydrogen peroxide caused conformational changes and, eventually, rendered the protein susceptible to degradation by the proteasome. We have mapped the specific methionine residues which are oxidized. Surface exposed methionines are initially oxidized,with retention of activity and resistance to degradation by the proteasome. Thus, these surface exposed residues act as the final oxidative defense which can prevent damage to the functionally critical regions of proteins.